The present invention encompasses a process for the enhancement of naphthalene dioxygenase activity in organisms that have been stably transformed with a DNA sequence encoding a multiple component naphthalene dioxygenase enzyme. In particular, the enhancement of naphthalene dioxygenase activity is evident in a microbe transformed with multiple copies of the ferredoxin gene, in a microbe cultured in the presence of iron, and in a microbe transformed with a site specifically mutated ferredoxin gene.
Indigo, or indigotin, occurs as a glucoside in many plants of Asia, the East Indies, Africa, and South America, and has been used throughout history to dye textiles with a lasting navy blue color. Before elucidation of the structure and the synthetic production of this compound, natural indigo was liberated from plants by protracted fermentation processes and then introduced into fabric as a soluble, colorless glycosylated derivative called indican. The soluble indican was easily hydrolyzed to glucose and indoxyl and, upon exposure to air, the indoxyl would oxidize to indigo, generating the dark blue pigment in the fibers of the fabric.
The chemical structure of indigo was deduced in 1883 and a commercially feasible manufacturing process was developed about 1887. All known successful industrial processes involve the air oxidation of indoxyl to indigo.
U.S. Pat. No. 4,520,103 to Ensley describes a process for transforming E. coli with a DNA expression vector that contains a gene for the expression of the multiple component enzyme, naphthalene dioxygenase, (NDO). The presence of this enzyme in E. coli results in the ability of the microbe to synthesize indigo when the cells are cultured in the presence of a source of indole. Ensley et al., Science, 222:167 (1983) proposes that in the NDO-mediated indole to indigo biosynthesis, cis-indole-2,3-dihydrodiol and indoxyl are likely intermediates.